688 VII. VITAMINS A 



been separated from the liver oil of the Japanese ishinagi (Stereolepis ishin- 

 agiy^'^^ and of the cod (Gadus viorrhua)^^''^^^ by the formation of an addition 

 product with maleic anhydride or by distillation in a molecular still. A 

 number of other methods have been employed for the demonstration of the 

 nature of vitamin A in such sources. Bacharach and Smith "^ found that 

 vitamin A in cod liver oil was largely insoluble in 92% ethanol ; they con- 

 sidered this as proof that, in cod liver oil, vitamin A is largely in the ester 

 form. Reti^^^ employed the method of partition between petroleum ether 

 and ethyl or methyl alcohol for demonstrating the presence of the ester; in 

 this manner he obtained positive results with the oils of a number of fishes, 

 chickens, and various mammals. The above investigator also states that 

 vitamin A is never found free under such conditions before saponification of 

 the liver oils. In 1937, Hickman, ^^''•^^^ using molecular distillation and the 

 technic of the elimination curve, concluded that the vitamin A in both cod 

 and halibut liver oils is largel}^ esterified. In an extension of this work, 

 Gray, Hickman, and Brown ^^^ were able to show that rat livers contained 

 vitamin A almost entirely in the form of the ester, irrespective of whether 

 U.S.P. reference oil, vitamin A caproate, ^dtamin A stearate, distilled ester 

 concentrate, or (8-carotene was fed. In all cases there was a small amount 

 of free vitamin A alcohol. Reed and co-workers,"^ by the use of an en- 

 tirely independent technic — chromatographic adsorption — obtained re- 

 sults leading to the conclusion that, in halibut oil, 94% of the vitamin A ap- 

 pears as the ester, while in the commercial distilled palmitate 98% occurs as 

 the ester. 



However, Sobotka, Kann, and Winternitz,"^ who used the fluorescence 

 technic, came to conclusions, as regards the proportion of vitamin A occur- 

 ring as an ester, which diverged somewhat from those reported in the earlier 

 investigations. Their estimations of the proportion of ester and alcohol 

 were based upon the fact that the esters of vitamin A show a strong fluores- 

 cence in ethanol, M'hile the vitamin A alcohol gives only a weak fluorescence. 

 It was reported that, when this method is applied to U.S.P. reference oil, 

 halibut liver oil and oleum percomorphum, only 60% of the total vitamin 

 A appears to be in ester form, while 80% is present in combined form in the 

 commercial distilled vitamin A ester. 



The whole subject has been thoroughly re-examined by Kascher and Bax- 

 ter"^ by the use of several procedures. One of the most effective was a 



11" K. C. D. Hickman, Ind. Eng. Chem., 29, 1107-1111 (1937). 



115 K. C. D. Hickman, Ind. Eng. Chem., 29, 968-975 (1937). 



116 E. Le B. Gray, K. C. D. Hickman, and E. F. Brown, /. Nutrition, 19, 39-46 (1940). 

 "7 G. Reed, E. C. Wise, and R. J. L. Frundt, Ind. Eng. Chem., Anal. Ed., 16, 509-510 



(1944). 



118 H. Sobotka, S. Kann, and W. Winternitz, /. Biol. Chem., 152, 635-639 (1944). 



119 H. M. Kascher and J. G. Baxter, Ind. Eng. Chem., Anal. Ed., 17, 499-503 (1945). 



